Spinach (Spinacia oleracea L.) is a flowering vegetable plant belonging to the family Amaranthaceae. It is native to Southwestern and Central Asia, but today spinach is cultivated worldwide, mainly in temperate regions. The edible part of the spinach plant is a compact rosette shape of leaves attached to a short stem. The leaves are produced during the first stage of the plant's life cycle, the vegetative rosette stage. The second stage is the flowering stage, or the bolting stage. When bolting occurs, there is growth of an elongated stalk with flowers growing from within the main stem of the plant. Once the plant has reached the bolting stage, it is no longer possible to harvest marketable leaves. The plant will allocate its resources to flowering instead of leaf production, which will ultimately cause the leaves to wither. Bolting is thus a very undesired trait in the production of leafy vegetables such as spinach. Therefore, slow bolting spinach varieties are very much preferred by growers in order to optimize yield.
There are three basic types of spinach, namely savoy, semi-savoy, and smooth. The savoy type spinach have dark green, crinkly and curly leaves. The semi-savoy type spinach is a hybrid variety with slightly crinkled leaves. Smooth type spinach has broad, smooth leaves.
The leaves of a spinach plant are usually sold loose, bunched, in pre-packaged bags, canned or frozen. Each of these products requires its own type of leaves. The smooth and some of the semi-savoy varieties are predominantly used in processed products such as canned or frozen spinach, while the semi-savoy and savoy types are especially used for the fresh market. Smooth leaf varieties are better suited for processing purposes than semi-savoy and savoy varieties, because they are easier to clean and prepare for canning and freezing. Savoy leaf varieties, on the other hand, are preferred in the fresh market because the leaves pack looser than the smooth leaf varieties and are less likely to wilt or turn yellow.
Spinach plants are cultivated for their highly nutritious leaves. The leaves are an extremely good source of vitamins such as A, B6, C, E, K and folate, and minerals such as calcium, iron, magnesium and potassium. Green leafy vegetables such as spinach are also rich in carotenoids such as lutein and beta-carotene, which have highly desirable antioxidant properties. Aside from their health properties, carotenoids are also pigmented molecules and play an important role in photosynthesis. In the photosynthetic pathway, carotenoids absorb light energy and transfer it to chlorophyll, which then uses the energy for photosynthesis.
In plants, chlorophyll is a pigmented molecule that lends to the green color in leaves. There is evidence that suggests that the amount of chlorophyll a, chlorophyll b, and total chlorophyll in green leafy vegetables is highly correlated with lutein and beta-carotene, suggesting that carotenoid levels may indirectly be determined using chlorophyll concentration (Mou, B. Journal of the American Society for Horticultural Science Vol 130 pp. 870-876, 2005, incorporated herein by reference). Therefore, dark green spinach with high chlorophyll content, and correspondingly high amounts of lutein and beta-carotene, are attractive to consumers for their potentially natural health properties.
Downy mildew is probably the most widespread and potentially destructive global disease of spinach. The causal agent of downy mildew on various Amaranthaceae, including spinach, is regarded as a single species, Peronospora farinosa. In particular, Peronospora farinosa f. sp. spinaciae infects spinach. Initial symptoms of downy mildew consist of dull to bright yellow necrotic lesions that appear on the leaves of infected spinach plants. With time the lesions can enlarge and become tan and dry. The infection can spread very rapidly, and it can occur both in glasshouse cultivation and in soil cultivation, resulting in widespread crop damage. In addition to the loss of quality of the leaves due to the lesions, downy mildew can also cause breakdown and rot of the infected leaves if they are packaged in bags or cartons.
The optimal temperature for formation and germination of P. farinosa f. sp. spinaciae spores is 9 to 12° C., and it is facilitated by a high relative humidity. When spores are deposited on a humid leaf surface they can readily germinate and infect the leaf. Fungal growth is optimal between 8 and 20° C. and a relative humidity of ≧80%, and within 6 and 13 days after infection mycelium growth can be observed. Oospores of P. farinosa can survive in the soil for up to 3 years, or as mycelium in seeds or living plants. Although some fungicide treatments may be effective, they can be costly and may raise ecological or pollution concerns, and may not be desired by organic farmers.
In recent years various resistance genes have been identified that provide spinach plants with a resistance against downy mildew. However, it has been observed that previously resistant spinach cultivars can again become susceptible to the fungus. Investigations revealed that the cultivars themselves had not changed, and that the loss of downy mildew resistance must therefore be due to P. farinosa overcoming the resistance in these spinach cultivars. The downy mildew races (also called physios, isolates, or strains) that were able to infect resistant spinach cultivars were collected in a differential reference set, which can be used to test spinach cultivars for resistance. The differential set may comprise a series of spinach cultivars (hybrids) that have different resistance patterns to the currently identified pathogenic races.
Currently there are 13 officially recognized races of Peronospora farinosa f. sp. spinaciae, designated races pfs1 to pfs13 (Irish et al. Phytopathol. Vol. 98 number pg. 894-900, 2008; Plantum NL (Dutch association for breeding, tissue culture, production and trade of seed and young plants) press release, “Denomination of Pfs: 12, a new race of downy mildew in spinach”, Feb. 28, 2011; Plantum NL (Dutch association for breeding, tissue culture, production and trade of see and young plants) press release, “Benoeming van fysio Pfs: 13, een nieuwe fysio van valse meeldauw in spinazie”, Aug. 2, 2011.
Newly identified Peronospora races can break the resistance of many spinach varieties that are currently used commercially worldwide, and they thus pose a serious threat to the productivity of the spinach industry. For this reason new resistance genes are very valuable assets. In order to confer a resistance that is as broad as possible, i.e. that confers resistance to as many Pfs races as possible, preferable to all known Pfs races, it is very useful to be able to stack different resistance genes against Peronospora infection in spinach. This is achieved by stacking various resistance genes which have overlapping resistance patterns. In this way it becomes more difficult for the pathogen to overcome the resistance.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.